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BOR - Papers in Press, published online ahead of print January 12, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.037598
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BIOLOGY OF REPRODUCTION 72, 1134–1141 (2005)
DOI: 10.1095/biolreprod.104.037598
© 2005 by the Society for the Study of Reproduction, Inc.

Changes in the Expression of Steroidogenic and Antioxidant Genes in the Mouse Corpus Luteum During Luteolysis1

Nastaran Foyouzi 3, Zailong Cai 3, Yukihiko Sugimoto 4, and Carlos Stocco 2 3

Department of Obstetrics, Gynecology, and Reproductive Science,3 Yale University School of Medicine, New Haven, Connecticut 06520 Department of Physiological Chemistry,4 Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Luteal cell death plays a key role in the regulation of the reproductive process in all mammals. It is also known that prostaglandin (PG) F2{alpha} is one of the main factors that cause luteal demise; still, the effects of PGF2{alpha} on luteal gene transcription have not been fully explored. Using microarray and reverse transcription-polymerase chain reaction, we have profiled gene expression in the corpus luteum (CL) of wild-type and PGF2{alpha} receptor knockout mice on Day 19 of pregnancy. Western blot analysis of selected genes was also performed. Because luteolysis has been shown to be associated with increased oxygen radical production and decreased progesterone synthesis, we report here changes observed in the expression of antioxidant and steroidogenic genes. We found that luteal cells express all genes necessary for progesterone synthesis, whether or not they had undergone luteolysis; however, an increase in mRNA levels of enzymes involved in androgen production, along with a decrease in the expression of enzymes implicated in estrogen synthesis, was observed. We also identified six genes committed to the elimination of free radical species that are dramatically down-regulated in the CL of wild-type animals with respect to PGF2{alpha} receptor knockout mice. Similar changes in the expression of steroidogenic and antioxidant genes were found in the CL of wild-type animals between Days 15 and 19 of pregnancy. It is proposed that an increase in the androgen:estrogen biosynthesis ratio, along with a significantly reduced expression of free radical scavenger proteins, may play an important role in the luteolytic process.

corpus luteum function, female reproductive tract, ovary, pregnancy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Luteal cell death plays a key role in the regulation of the reproductive process in all mammals. Because of the negative effect of luteal progesterone on the secretion of gonadotropin hormones, the demise of the corpus luteum (CL) is essential for the initiation of a new reproductive cycle. In species such as rats, mice, and goats, in which the CL is the only source of progesterone throughout pregnancy, luteolysis is also necessary for parturition to occur. Prostaglandin (PG) F2{alpha} plays a key role in the initiation of labor and parturition in rodents, and it is vital for the induction of the luteolytic process. The luteolytic effect of PGF2{alpha} was first reported in 1969 by Pharriss and Wyngarden [1]. Later in the last century, it was demonstrated that mice rendered deficient for the PGF2{alpha} receptor gene do not show the normal prepartum drop in progesterone and, as a consequence, do not give birth [2]. Despite this dramatic phenotype, PGF2{alpha} receptor knockout mice have a normal gestation, and normal fetuses can be rescued by cesarean delivery or ovariectomy, indicating that the primary role of PGF2{alpha} is limited to the days before parturition, and its principal target is the ovary [2]. Subsequently, researchers demonstrated that the CL of PGF2{alpha} receptor knockout mice fails to express the enzyme 20{alpha}-hydroxysteroid dehydrogenase (E.C. 1.1.1.149) at the end of pregnancy, resulting in sustained levels of progesterone in circulation [3]. This evidence indicates that, in rodents, the CL is the main ovarian structure affected by PGF2{alpha} at the end of pregnancy. Whether the expression of other steroidogenic enzymes besides 20{alpha}-hydroxysteroid dehydrogenase is affected by the lack of PGF2{alpha} receptor expression is not known.

It is now firmly established that the CL produces significant amounts of reactive oxygen species (ROS) at regression. It has been shown that hydrogen peroxide and lipid peroxides are generated during luteolysis associated with ascorbic acid and {alpha}-tocopherol depletion [4–6]. There is an increase in superoxide anion production in luteal membranes after challenge in vivo with PGF2{alpha} [7], or in isolated luteal cells treated with PGF2{alpha} [8–10]. However, whether ROS mediate the action of PGF2{alpha} or are a consequence of luteal regression is an unresolved question. Similarly, it is not known whether changes in the expression of genes involved in the metabolism of ROS take place during the luteolytic process.

The mechanisms by which PGF2{alpha} affects luteal function have been extensively studied and reviewed [11]; however, the effects of this hormone on luteal gene transcription at the end of pregnancy in rodents have only lately begun to be studied. For instance, although early experiments clearly established that PGF2{alpha} increases 20{alpha}-hydroxysteroid dehydrogenase activity [12], the discovery that this effect is due to the stimulation of the Akr1c18 gene was only recently published [3, 13]. A gene expression profile of the rat CL has been previously reported [13]. In that report, rats on Day 19 of pregnancy were treated with PGF2{alpha} or vehicle, and the expression of several genes was accessed 24 h afterward. Although this is a valid model in which to study the effect of PGF2{alpha} on luteal gene expression, it does not represent a physiological situation, and questions are raised regarding dose and time of treatment. In addition, only a limited number of genes directly involved in luteal function were included. As mentioned before, gestation proceeds without any alteration in PGF2{alpha} receptor knockout mice, suggesting normal luteal function. Strikingly, the CL of these animals fails to undergo luteolysis, and luteal function persists beyond the normal period of pregnancy. The aim of this investigation was to profile gene expression in the CL in mice of the wild type and in PGF2{alpha} receptor knockout mice on Day 19 of pregnancy. In this model, the genomic effects of physiological endogenous levels of PGF2{alpha} were studied at the time when the normal luteolytic process takes place. We report here changes observed in the expression of genes involved in steroidogenesis and ROS metabolism.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

PGF2{alpha} receptor knockout mice with a mixed genetic background of 129/Ola and C57BL/6 strains were used [2]. Wild-type and PGF2{alpha} receptor knockout mice were maintained at 23°C under a 12L:12D cycle. Virgin females (9 to 12 wk of age) housed overnight with males were checked the following morning for vaginal plugs. The day the plug was found was counted as Day 1 of pregnancy. Wild-type animals were killed in the morning of Days 15 or 19 of pregnancy, whereas CL of PGF2{alpha} receptor knockout mice were obtained only in the morning of Day 19 of pregnancy. Animal care and handling conformed to the National Institutes of Health guidelines for animal research. The experimental protocol was approved by the Yale University Animal Resources Center.

Affymetrix Gene Chip Analysis

We used the mouse 430 2.0 chip array from Affymetrix (Santa Clara, CA). Total RNA CL was isolated using TRIzol reagent and further purified using the RNeasy kit (Qiagen, Valencia, CA). The quality of total RNA was evaluated by A260/A280 ratio and by electrophoresis. Preparation of labeled cRNA for hybridization onto Affymetrix GeneChips was performed following the recommended Affymetrix protocol. Double-stranded cDNA was synthesized from 6 µg of total RNA by using the Superscript Choice System (Invitrogen, Carlsbad, CA), with a high performance liquid chromatography-purified oligomer (dT) 24 primer containing a T7 RNA polymerase promoter sequence (Genset, La Jolla, CA). The second cDNA strand was synthesized through the use of Escherichia coli DNA polymerase I, RNase H, and DNA ligase. The cDNA was precipitated with ethanol and resuspended in RNase-free water. Labeled cRNA was generated from cDNA by in vitro transcription using a Bioarray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics, Farmingdale, NY), following the manufacturer's instructions and incorporating Cy5-UTP. Labeled cRNA was purified using an RNeasy column before fragmenting to a size of 35–200 bases by incubating at 94°C for 35 min in fragmentation buffer (40 µM Tris-acetate pH 8.1, 100 µM potassium acetate, 30 µM magnesium acetate). The arrays were hybridized for 16 h at 45°C. After being hybridized, the arrays were washed using the fluidics station (Affymetrix) and scanned on a GeneChip Scanner 3000 (Affymetrix). Expression analysis of all experiments was performed with GeneSpring 5.1 (Silicon Genetics, Redwood City, CA) using analysis data generated by Microarray Suite software. (Affymetrix, Santa Clara, CA) Differential expression was defined as those transcripts that had a difference of 2-fold or more. Performed with GeneSpring software, the cross-gene-error model was used in combination with a one-way analysis of variance (ANOVA) (significance level set at P < 0.05).

Relative Reverse Transcription-Polymerase Chain Reaction Analysis

Total RNA from frozen mouse CL was isolated using Tri-Reagent (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. One microgram of total RNA was reverse-transcribed at 42°C with the Advantage RT-for-PCR kit (Promega, Madison, WI) and later diluted to a final volume of 100 µl. The polymerase chain reaction (PCR) mixture containing specific oligonucleotide primers (500 pmol), deoxyribonucleotide triphosphate (150 µM) and Ex Taq DNA polymerase (1 unit; Takara, Kyoto, Japan) was added to each tube containing 5 µl of reverse transcription (RT) product. Each PCR reaction also included ß-actin primers used as internal controls. Before proceeding with the semiquantitative PCR, conditions were established such that the amplification of the products was in the exponential phase, and the assay was linear with respect to the amount of input cDNA. Band intensity was determined by using ImageJ software from the National Institutes of Health (Bethesda, MD). Each experiment was repeated three times. A Student t-test was used to determine whether differences between groups were statistically different.

Western Blotting Analysis

Corpora lutea from wild-type mice on Days 15 and 19 of pregnancy and from PGF2{alpha} receptor knockout mice were homogenized in ice-cold lysis buffer (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Nonidet p-40, 0.5% sodium deoxycholate, 0.1% SDS, 40 µM PMSF, 0.3 µM aprotinin, and 1 µM leupeptin). This was followed by a 30-min incubation on ice and centrifugation at 10 000 x g for 20 min at 4°C. The supernatant was transferred to new tubes, aliquoted, and stored at –70°C until the time of electrophoresis. An aliquot of the supernatant was kept for protein measurement, using BSA as a standard. Samples were denatured by adding a sample buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 0.01% bromophenol blue), followed by boiling for 10 min. Thirty micrograms of protein were separated on 10% SDS-PAGE gels in Tris-glycine 0.1% SDS buffer; and transferred to nitrocellulose paper in 25 mM Tris, 192 mM glycine, and 20% methanol buffer at 250 mA for 1.5 h. Blots were incubated for 2 h at room temperature in 5% nonfat dry milk to block unspecific binding. Blots were then washed and incubated overnight at 4°C with anti-P450 aromatase antibody (Serotech, U.K.) at a 1/4000 dilution, with antisuperoxide dismutase 2 antibody (Calbiochem, San Diego, CA) at a 1/800 dilution or with anti-ß-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a 1/1000 dilution. After three washes, blots were incubated with a corresponding secondary antibody conjugated to horseradish peroxidase (1/20 000 dilution) for 2 h at room temperature. Protein-antibody complexes were visualized using Western Blotting Luminol Reagent, following the manufacturer's protocol (Santa Cruz Biotechnology).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Global Analysis of Gene Expression in Mouse CL on Day 19 of Pregnancy

Probes derived from total RNA extracted from CL of wild-type or PGF2{alpha} receptor knockout mice were hybridized to the mouse 430 2.0 chip array from Affymetrix. A total of four pairs of samples and comparisons were performed. Figure 1 shows a representative distribution of the intensity of all genes present in the microarray. The average Pearson correlation coefficient was 0.929, indicating similar levels of expression between samples. A total of 1373 genes demonstrated at least a 2-fold change in expression. The statistical significance of these changes was determined by ANOVA included in the GeneSpring software. Only genes with a net intensity equal to or higher than 10 times the background were chosen for further analysis. Between these highly expressed genes, 169 were down-regulated and 101 were up-regulated in the CL of PGF2{alpha} receptor knockout mice. Of these genes, 11 are involved in steroid synthesis and ROS metabolism. To provide a more complete analysis, we report the expression of those genes that were significantly affected by the lack of PGF2{alpha} receptor expression as well as the expression of several key genes involved in luteal steroidogenesis and ROS metabolism.



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  		FIG. 1. Expression profiling of 45k genes between the CL of wild-type and PGF2{alpha} receptor knockout mice. For each gene, average expression levels (in arbitrary units) were calculated from four independent experiments and displayed on a scatter plot. *Pearson correlation coefficient + SEM, n = 4

Expression Levels of Steroidogenic Genes on Day 19 of Pregnancy in CL of Wild-Type or PGF2{alpha} Receptor Knockout Mice

To facilitate the interpretation and integration of the results, a simplified scheme of rat luteal steroidogenesis is presented in Figure 2A. As shown in Figure 2B (top), no changes in mRNA levels of the scavenger receptor, class B, type 1 (Scarb1), the steroidogenic acute regulatory (Star), and the benzodiazepine receptor peripheral-type (Bzrp) were found between knockout and wild-type animals. Analysis of the relative expression levels of these genes shows high relative expression of Scarb1 and Star, both in wild-type and PGF2{alpha} receptor knockout mice (Fig. 2A, lower). Messenger RNA relative expression of Bzrp was 10 times lower than Star relative expression.



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  		FIG. 2. A) A simplified scheme of the cholesterol and steroid metabolism in mouse luteal cells. B) The top graph represents the ratio of the luteal expression of steroidogenic genes in wild-type and PGF2{alpha} receptor knockout mice. The scale indicates the values of expression as a ratio between wild-type and PGF2{alpha} receptor knockout animals (higher expression in wild type) or –1 x PGF2{alpha} receptor knockout:wild-type for negative values (higher expression in knockout). The bottom graph reports the relative expression of these genes against the housekeeping gene ß-actin. Bars represent the average of four different experiments + SEM. *Genes whose expression is significantly different between wild-type and PGF2{alpha} receptor knockout mice (P < 0.05; one-way ANOVA)

A significant increase (3.5-fold) in mRNA levels of the enzyme cholesterol 25-hydroxylase (Ch25h) was found in wild-type animals compared with that in PGF2{alpha} receptor knockout mice. No differences were found between wild-type and PGF2{alpha} receptor knockout mice in the expression of cytochrome P450scc (Cyp11a1), nor of 3ß-hydroxysteroid dehydrogenase {Delta}4{Delta}5 isomerase (Hsd3b) types 1 and 7 (Fig. 2B, top). Cyp11a1 and Hsd3b1 were found to be the most highly expressed genes, not only between the steroidogenic genes (Fig. 2B, lower), but also among all the genes present in the microarray used (not shown). No expression of other Hsd3b mRNA isoforms was found. As previously reported, a significant increase in 20{alpha}-hydroxysteroid dehydrogenase (Akr1c18) mRNA levels was found in wild-type animals (Fig. 2B).

Significant differences in P45017{alpha}-hydroxylase/C17–20 lyase (Cyp17a1) and cytochrome P450 aromatase (Cyp19a1) mRNA levels were found between PGF2{alpha} receptor knockout mice and wild-type animals (Fig. 2B, top). High mRNA levels of Cyp19a1 were found in PGF2{alpha} receptor knockout mice, whereas Cyp17a1 mRNA levels were higher in wild-type mice.

The chip array we used contains probes for all known 17ß-hydroxysteroid dehydrogenase (Hsd17b) isoforms; however, only Hsd17b types 1, 4, and 7 mRNA were found to be expressed in the CL. Hsd17b1 and Hsd17b4 relative expressions were low, and no differences were observed between wild-type and PGF2{alpha} receptor knockout mice (Fig. 2B, lower). Of interest, Hsd17b7 mRNA levels were significantly higher in PGF2{alpha} receptor knockout mice compared with levels in wild-type animals (Fig. 2B, top).

Luteal Antioxidant Capability in CL of Wild-Type or PGF2{alpha} Receptor Knockout Mice on Day 19 of Pregnancy

A simplified scheme of oxygen radical metabolism in presented in Figure 3A. As shown in Figure 3B, it was found that the mouse CL expresses mRNA for all superoxide dismutase (SOD) enzymes (i.e., Sod1, Sod2, and Sod3; soluble or copper-zinc, mitochondrial or manganese, and extracellular, respectively). Sod2 was found to be the most highly expressed (Fig. 3B, lower), and its mRNA levels were significantly lower in wild-type animals compared with levels found in PGF2{alpha} receptor knockout mice (Fig. 3B, top). Similarly, a decrease of 4.4-fold in Sod3 mRNA levels in wild-type animals was observed compared with levels in knockout animals, whereas mRNA levels of Sod1 were no different between these groups. Of interest, mRNA levels of a protein called copper chaperone for SOD1 (Ccs), were significantly lower in wild-type animals.



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  		FIG. 3. A) A simplified scheme of ROS metabolism. The high dot (·) indicates the free radical. B) The top graph represents the negative ratio of the luteal expression of genes involved in ROS metabolism in PGF2{alpha} receptor knockout and wild-type animals. The bottom graph reports the relative expression of these genes against the housekeeping gene ß-actin. Bars represent the average of four different experiments + SEM. *Genes whose expression is significantly different between wild-type and PGF2{alpha} receptor knockout mice (P < 0.05; one-way ANOVA)

The results also showed that the mRNA of all members of the peroxiredoxin (Prdx) family of proteins are expressed in the CL. Prdx1, Prdx2, Prdx3, and Prdx6 mRNA were found to be highly expressed, while Prdx4 and Prdx5 were found to be present at low levels (Fig. 3B, lower). Low relative expression levels of catalase (Cat) were found (Fig. 3, lower). Messenger RNA levels of Prdx6 were significantly lower in wild-type mice compared with PGF2{alpha} receptor knockout animals (Fig. 3B, top).

High luteal mRNA levels for glutathione peroxidase (Gpx) types 1, 3, and 4 were found in both wild-type and PGF2{alpha} receptor knockout mice (Fig. 3B, lower), and no differences were found in mRNA levels of these enzymes between these two groups (Fig. 3B, top).

It was also found that luteal cells express the mRNA for the {alpha}-tocopherol transfer protein (Ttpa). Levels of Ttpa mRNA were 10-fold higher in luteal cells of PGF2{alpha} receptor knockout mice than in cells of wild-type animals (Fig. 3B). In wild-type mice, a significant decrease in microsomal glutathione S-transferase (Mgst) 2 mRNA levels was found compared with levels in PGF2{alpha} receptor knockout mice, but no differences were found in the expression of Mgst1 and Mgst3 (Fig. 3B, top). Mgst1 mRNA was found to be expressed at very high relative levels, whereas Mgst2 and Mgst3 were expressed at moderate and low relative levels, respectively.

Validation of Microarray Results

To validate our microarray data, semiquantitative RT-PCR was used. To control for mRNA recovery and RT efficiency, all RT-PCR experiments were normalized to the housekeeping gene ß-actin. RT-PCR data were consistent with microarray results; thus, relative mRNA levels of Ch25h, Akr1c18, and Cyp17a1 were significantly higher in wild-type animals. Luteal Cyp19a1 and Hsd17b7 relative mRNA levels were significantly higher in PGF2{alpha} receptor knockout mice (Fig. 4A). Relative mRNA levels for Sod2, Sod3, Ccs, Pdrx6, Mgst2, and Ttpa were significantly up-regulated in PGF2{alpha} receptor knockout mice (Fig. 4B). Fold changes observed by RT-PCR were also consistent with those observed in the microarray experiment. No significant changes in ß-actin (Actb) mRNA levels were found between samples.



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  		FIG. 4. Reverse transcription analysis of steroidogenic genes (A) and genes involved in ROS metabolism (B) that were found to be significantly different between wild-type (+/+) and PGF2{alpha} receptor knockout (–/–) mice. The relative expression columns on the left represent the densitometric analysis of each band, expressed as a ratio between the intensity of each gene and ß-actin (average and SEM, n = 4). *P < 0.05; **P < 0.01 (Student t-test). The ratio column represents the ratio between the relative intensity of each gene on –/– and +/+ animals

Changes in Luteal Gene Expression Between Days 15 and 19 of Pregnancy in Wild-Type Animals

Using microarray analysis, we identified several genes that are differentially expressed in the CL of PGF2{alpha} receptor knockout and wild-type animals. To further extend these studies and to confirm the participation of these genes in the luteolytic process, we examined their relative expression in the CL of wild-type animals on Day 15, which is a fully functional CL, and CL that had already initiated the luteolytic process obtained from mice on Day 19 of gestation.

Relative mRNA levels for Ch25h were significantly higher on Day 19 of pregnancy than on Day 15. The opposite scenario was found when relative mRNA levels for Cyp19a1 and Hsd17b7 were studied. Cyp17a1 mRNA was not detectable on Day 15 of pregnancy, but high levels were found on Day 19 (Fig. 5).



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  		FIG. 5. Expression of steroidogenic and antioxidant genes in wild-type mouse CL on Day 15 and Day 19 of pregnancy. The relative expression columns on the left represent the densitometric analysis of each band, expressed as a ratio between the intensity of each gene and ß-actin (average and SEM, n = 3). *P < 0.05; **P < 0.01 (Student t-test). The ratio column represents the ratio between the relative intensity of each gene on Days 15 and 19 of pregnancy

Higher and statistically significant relative mRNA levels for Sod2, Ccs, Prdx6, Mgst2, and Ttpa were observed on Day 15 of gestation compared with Day 19 of gestation (Fig. 5). Lower but statistically nonsignificant Sod3 mRNA levels were observed on Day 19 of pregnancy with respect to Day 15. No changes in Actb mRNA levels were found between samples.

Western Blot Analysis for Cytochrome P450 Aromatase (CYP19A1) and SOD2 Protein Levels in Wild-Type Animals on Days 15 and 19, and in PGF2{alpha} Receptor Knockout Mice on Day 19 of Gestation

Next, we used Western Blot to investigate whether changes in mRNA levels of Cyp19a1 and Sod2 are reflected in variations in their protein levels. As shown in Figure 6, luteal levels of CYP19A1 and SOD2 were low in wild-type mice on Day 19 of pregnancy, whereas high levels were found in wild-type animals on Day 15 and in PGF2{alpha} receptor knockout mice on Day 19 of gestation. No changes in ß-actin protein (ACTB) levels were found between samples.



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  		FIG. 6. Cytochrome P450 aromatase (CYP19A1), superoxide dismutase 2 (SOD2), and ß-actin (ACTB) protein expression levels of wild-type (+/ +) animals on Days 15 and 19 of pregnancy and of PGF2{alpha} receptor knockout (–/–) mice on Day 19 of gestation. Whole corpora lutea proteins were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with specific CYP19A1, SOD2, or ACTB antiserum. Blots are representative of two different sets


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results indicate that during the normal luteolytic process that takes place at the end of pregnancy in rodents, profound changes occur in luteal expression of androgen and estrogen synthetic enzymes, in addition to the well-known increase in progesterone metabolism. It is likewise interesting that several genes involved in the elimination of free radical species are dramatically down-regulated in luteal cells undergoing luteolysis.

Steroidogenic Genes

The results presented clearly indicate that no changes in luteal mRNA levels of enzymes involved in the uptake of cholesterol or in the synthesis of progesterone take place during luteolysis in mice (Fig. 2A). Accordingly, we have found high relative mRNA levels of the scavenger receptor, class B, type 1 (Scarb1) both in wild-type and PGF2{alpha} receptor knockout mice. SCARB1 binds high-density lipoproteins [14], which are the preferential source of cholesterol in the luteal cells of rodents [15–17], whereas steroidogenic acute regulatory (STAR) [18] and benzodiazepine receptor peripheral-type (BZRP) [19] proteins mediate the transport of cholesterol into the mitochondrion, which is a limiting step in steroid synthesis. We confirmed previous reports showing Star and Bzrp mRNA expression in the CL [20, 21]. In addition, we demonstrated that in the mouse CL Star mRNA expression levels are at least 10 times higher than those of Bzrp, and that the expression of these genes does not differ between wild-type and PGF2{alpha} receptor knockout mice.

There is evidence indicating that treatment of ewes or cows with PGF2{alpha} dramatically reduces concentrations of mRNA encoding Star [22, 23]. In pseudopregnant rats, PGF2{alpha} has also been shown to reduce Star luteal mRNA levels [21]. In contrast, our present results indicate that, in mice, PGF2{alpha} does not affect Star expression at the end of pregnancy. Direct and indirect evidence support our present finding. PGF2{alpha} administration to pregnant rats on Day 19 does not affect Star mRNA levels (unpublished results). Moreover, in mice and rats, total progestin production (progesterone plus 20{alpha}-DH-progesterone) remains constant after PGF2{alpha} treatment [3], indicating that cholesterol is still supplied without restriction to the cytochrome P450scc, further suggesting that luteal expression of Star was not affected by PGF2{alpha} treatment. This differential effect of PGF2{alpha} on Star expression is probably due to fundamental differences in the regulation of the CL between species and during pregnancy in rats. In ovine and bovine, LH is the main luteotropic hormone [24]. In rats, however, LH is necessary to sustain luteal function only between Days 7 and 10 of pregnancy [25] while placental lactogens are the main luteotropic hormones during the second part of pregnancy [26].

Of interest, we found an increase in the mRNA levels of the enzyme cholesterol 25-hydroxylase (Ch25h) in wild-type animals. The gene for this enzyme was first cloned in 1998 by Lund et al. [27], and the enzyme was shown to cause 25-hydroxylation of cholesterol. Although Ch25h is not highly expressed, 25-hydroxycholesterol is a potent regulatory oxysterol and suppresses the activation of transcription factors such as sterol regulatory element-binding proteins, leading to inhibition of gene transcription [27]. Whether 25-hydroxycholesterol plays any role in the regulation of luteal gene expression is unknown.

In the rat and mouse, a key role controlling progesterone secretion by the CL is played by 20{alpha}-hydroxysteroid dehydrogenase (AKR1C18), which converts progesterone into an inactive steroid, 20{alpha}-dihydroprogesterone [28, 29]. We confirmed the stimulatory effect of PGF2{alpha} on Akr1c18 expression [3] at the end of pregnancy. A novel finding of this report is that the effect of PGF2{alpha} is not limited to an increase in progesterone catabolism, but it also profoundly affects androgen and estrogen synthesis. P45017{alpha}-hydroxylase/C17–20 lyase (Cyp17a1) is highly expressed in the CL of pregnancy rats until Day 12, when it drops to undetectable levels [30]. In agreement with these results, Cyp17a1 mRNA was not detected on Day 15 of gestation. Of interest, we found high Cyp17a1 mRNA levels on Day 19 of pregnancy, suggesting that luteal expression of this enzyme increases again toward the end of pregnancy. In support of this finding, an increase in ovarian androgen production has been shown at this time [31]. We also observed that Cyp17a1 mRNA levels are significantly low in PGF2{alpha} receptor knockout mice compared with those in wild-type animals. Taken together, these findings suggest that luteal Cyp17a1 expression increases at the end of pregnancy, and that PGF2{alpha} action in the CL is required for this to occur. Because it has been demonstrated that administration of dihydrotestosterone, a nonaromatizable androgen, induces abortion [32], and that androstenedione reduces progesterone production in luteal cells [33, 34], we postulate that CYP17A1 may play an important role in the normal progress of luteolysis by increasing intraluteal levels of androstenedione.

Biosynthesis of estrone from androstenedione is catalyzed by cytochrome P450 aromatase (CYP19A1). In the rat, the luteal contents of Cyp19a1 mRNA is low on Day 4 of pregnancy, progressively increases reaching maximal levels between Days 15 and 19 of gestation, and rapidly decreases to almost undetectable levels on Day 23 [35, 36]. The mechanisms that control Cyp19a1 gene expression in luteal cells, particularly the one or more factors that cause its rapid fall before parturition, are not known. Because we have previously shown that PGF2{alpha} administration to pregnant rats on Day 19 decreases Cyp19a1 gene expression [37], and because our present results show high Cyp19a1 (mRNA) and CYP19A1 (protein) levels in PGF2{alpha} receptor knockout mice with respect to wild-type animals, we postulate that PGF2{alpha} is accountable for the dramatic decrease in Cyp19a1 expression that takes place in the CL of rodents at the end of pregnancy. Finally, not only synthesis of estrone, but also its conversion to the more active estrogen compound, 17ß-estradiol, seems to be regulated by PGF2{alpha} in luteal cells on Day 19 of pregnancy. In agreement with previous studies [38–41], we found that mRNA levels of 17ß-hydroxysteroid dehydrogenase (Hsd17b) types 1 and 7 are high in mouse CL. HSD17B1 and HSD17B7 transform estrone into 17ß-estradiol [40, 42]. It has been shown that luteal Hsd17b7 mRNA expression rapidly decreases at the end of pregnancy in the rat [43, 44] and mouse [40]. We observed that this decrease does not take place in PGF2{alpha} receptor knockout mice.

Antioxidant Genes

As a consequence of normal metabolism, cells generate oxygen radical species. In the particular case of luteal cells, an increase in the generation of ROS has been correlated with decreased progesterone production and cell death [5, 8, 10]. The first reaction of O2 is a one-electron reduction to superoxide (O2·–; Fig. 3A). The major defense mechanism against this radical is a family of enzymes named superoxide dismutase, which catalyze the dismutation of superoxide to H2O2. We have shown here that the CL of mice contains mRNA for SOD1, SOD2, and SOD3. Our results show that Sod2 mRNA is not only highly expressed, but that its expression is dramatically regulated during pregnancy. We found that in wild-type animals, Sod2 mRNA and SOD2 protein levels are high on Day 15 of pregnancy, but their levels of expression significantly decrease on Day 19. It is interesting that this decrease in Sod2 mRNA and SOD2 protein levels does not take place in PGF2{alpha} receptor knockout animals. Messenger RNA levels for SOD3 and a protein called copper chaperone for SOD1 (CCS) were also found to be affected by the lack of PGF2{alpha} receptor. CCS, by providing Cu(I) to SOD1, is essential for SOD1 activity [45–47]. Evidence suggests that SOD1 plays an important role in ovarian physiology. Thus, it has been shown that Sod1 mRNA antisense decreases luteal progesterone production [48], and that fertility is reduced in Sod1–/– mice [49]. However, the mechanisms that control Sod1 gene expression, or SOD1 activity (or both) in the CL remain to be determined. Our results suggest that luteal SOD1 activity may be regulated indirectly through a decrease in CCS protein expression. These results suggest that at the end of pregnancy, a decrease in SOD2, SOD3, and CCS expression significantly reduces the capacity of luteal cells to cope with superoxide accumulation. Furthermore, PGF2{alpha} action in the CL is necessary for the decrease on the expression of these enzymes to occur.

After conversion of O2·– to H2O2, the next step is the reduction of H2O2 to water. Several enzymes catalyze this reaction, including catalase, glutathione peroxidase (GPX), and a relative newly discovered family of antioxidant proteins, peroxiredoxins (PRDX) [50]. We have found that the mouse CL contains mRNA for each one of these enzymes. Noteworthy is that mRNA levels for catalase found in the CL at the end of pregnancy are low compared with those of the Prdx family, suggesting that PRDX, but not catalase, may play a more important role in the elimination of hydrogen peroxide in luteal cells. It has been recently postulated that PRDX6 eliminates H2O2 and reduces the number of hydroxyl radicals produced by the Fenton reaction and the subsequent tissue injury [51]. Consistent with this finding, Prdx–/– mice were shown to have more severe tissue damage and higher mortality rates than controls, even when catalase (CAT) or GPX proteins were normally expressed [51]. In addition, macrophages from the Prdx–/– mutants contain more H2O2 and are more susceptible to oxidant-induced cell death than macrophages of wild-type animals, indicating that PRDX6 functions in vivo as an antioxidant and nonredundantly with respect to other PRDX and antioxidant enzymes. We have found that Prdx6 mRNA levels are highly expressed in nonluteolytic CL (Day 15 wild-type and Day 19 PGF2{alpha} receptor knockout animals), suggesting that a drop in Prdx6 expression may be important for the normal evolution of the luteolytic process. To our knowledge, this is the first report on the expression and regulation of Prdx6 mRNA levels in luteal cells.

The hydroxyl radical is the most reactive free radical [52]. It could be formed in vivo from superoxide, or from hydrogen peroxide via the Fenton reaction (or both) (Fig. 3A). Hydroxyl radicals immediately react with surrounding target molecules at the site where they are generated, especially with polyunsaturated fatty acid-producing lipid hydroperoxides (L-OO·). Lipid hydroperoxides can then react with other lipids, propagating their own formation. This chain reaction leads to membrane damage. Protection against lipid peroxidation is given in part by {alpha}-tocopherol, which is highly concentrated in luteal cells [8, 53]. Reduced {alpha}-tocopherol is then regenerated by ascorbic acid. We have found that luteal cells express the mRNA for {alpha}-tocopherol transfer protein (TTPA), which is a cytosolic protein that in the liver selectively transfers {alpha}-tocopherol from lipoproteins taken up by hepatocytes via the endocytic pathway to newly secreted lipoproteins [54]. Of interest, luteal cells undergoing luteolysis contain significantly lower levels of Ttpa mRNA levels than functional luteal cells, suggesting that TTPA may play an important role in the maintenance of normal luteal function; probably TTPA facilitates the accumulation of {alpha}-tocopherol in luteal membranes. Microsomal glutathione S-transferase 2 (MGST2) is also involved in scavenging lipid soluble radicals [55, 56]. MGST2 location, bound to the membrane of the endoplasmic reticulum where several steroidogenic enzymes are localized, suggests that lipid peroxides formed because the high rate of steroid synthesis in luteal cells may be efficiently eliminated by MGST2. Our results indicate that a decrease in the expression of this enzyme may be important during the luteolytic process in rodents.

As mentioned earlier, it is well known that hydrogen peroxide, superoxide anion, and lipid peroxides are generated during the luteolytic process [4–10], however, the events that lead to this accumulation of ROS are largely unknown. Our results have shed light on a possible mechanism by which this increase in ROS production takes place during luteolysis. It is proposed that PGF2{alpha} by decreasing the expression of free radical scavenger proteins, facilitates the accumulation in ROS, which in turn initiates a cascade of events that will finally cause luteal cell death.


   ACKNOWLEDGMENTS
 
We are thankful to Dr. Harold Behrman for his critical reading of the manuscript and to Dr. Se-Te Joseph Huang for his assistance in the analysis of microarray data.


   FOOTNOTES
 
1 Supported by a start-up grant from the Obstetrics, Gynecology, and Reproductive Science Department, Yale University School of Medicine. Back

2 Correspondence: Carlos Stocco, Yale University School of Medicine, 333 Cedar Street, P.O. Box 208063, New Haven, CT 06520. FAX: 203 785 7134; carlos.stocco{at}yale.edu Back

Received: 11 November 2004.

First decision: 16 December 2004.

Accepted: 23 December 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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